Rotating culture vessel and automatic cell culture apparatus using same

ABSTRACT

Disclosed is a rotating culture vessel based on a rotating culture technology using an RWV, by which cell seeding, liquid medium exchange, quality control and so on can be automated and degassing can be conducted simultaneously with liquid medium exchange without disturbing the cells under culture. Also disclosed is an automatic cell culture apparatus using the same. A rotating culture vessel, which contains cells and a liquid culture medium, to be attached to a horizontal rotating shaft of a rotating culture device to three-dimensionally culture the cells, wherein one or more inlets/outlets for supplying cells and a liquid culture medium at the early stage and then taking out the cultured cells, are formed at appropriate position of a flat cylindrical culture container; at least one pair of a supply port and a discharge port for liquid medium exchange is provided on the outer circumferential cylindrical face of the culture container.

TECHNICAL FIELD

The present invention relates to a rotating culture vessel and anautomatic cell culture apparatus using the vessel, and more specificallyrelates to a rotating culture vessel suited for automatic exchange of aliquid culture medium and an automatic cell culture apparatus having anautomatic liquid culture medium exchange function.

BACKGROUND ART

Conventionally, various automatic cell culture apparatuses have beensupplied. For example, Patent Document 1 pertaining to a priorapplication of the present applicants discloses an automatic cellculture system. The automatic cell culture system can handle a pluralityof culture cassettes containing a culture dish, automatically transferthe culture cassette with a robot arm for a dispensing operation and thelike, control atmospheres such as gas concentration, temperature, andhumidity in an incubator, and reliably prevent not only contamination inthe incubator but also contamination during transfer of the culture dishfor a dispensing operation and the like.

Patent Documents 2 and 3 also disclose an automatic cell cultureapparatus that automates, for example, an exchange operation of a liquidculture medium with a general purpose multiple joints type robot in atwo-dimensional culture apparatus. The automatic cell culture apparatushas an effect on the prevention of bacterial invasion by minimization ofhuman intervention, but an expensive general purpose industrial robotperforms the same operations as those that a human has performed, andsuch an apparatus requires high cost. Moreover, such an apparatus is notbeyond common two-dimensional cell culture apparatuses and is not anapparatus for achieving three-dimensional culture.

Meanwhile, mesenchymal stem cells derived from the bone marrow hasmultipotency to be differentiated into tissues such as bone, cartilage,adipose, and ligament. However, it is known that, in a common in vitroculture, the cells are settled to the bottom of a petri dish due to theEarth's gravity to form a two-dimensional sheet and the original cellcharacteristics disappear. To address this, a culture method using arotating wall vessel (RWV) bioreactor has been developed in order toachieve the three-dimensional culture in a microgravity environment thatis nearly gravity-free, and cells are three-dimensionally cultured withappropriate differentiation inducing factors to be differentiated intoan original tissue of the cell. A culture apparatus using the RWV isreleased from Synthecon. The apparatus is composed of a flat cylindricalvessel having a gas permeable membrane on the back side, and the vesselis rotated around an attaching part to which a horizontal rotating shaftof a rotation controller is attached. The rotation of the vesselconstantly changes the gravity direction to cells, and consequently cangenerate a microgravity environment having one-hundredth the groundgravity by time average. Hence, clumps of cells are not settled and canbe cultured while floating. The apparatus is typically referred to as arotating culture device.

The RWV used in the conventional rotating culture device includes aninlet/outlet for a cell suspension and a pair of a supply port and adischarge port for a liquid culture medium on the front face orthogonalto the rotating shaft, and the liquid culture medium is exchangedmanually. That is, for liquid culture medium exchange, the RWV isremoved from the rotating shaft and left with the supply port and thedischarge port facing upward, a rubber cap of the supply port is takenoff, a leading end of a supply syringe including a new liquid culturemedium is closely connected to the supply port, while a leading end ofan empty discharge syringe is closely connected to the discharge port,then cocks of the supply port and the discharge port are unlocked, apiston of the supply syringe is pushed by one hand to supply the liquidculture medium into the vessel, and simultaneously a piston of thedischarge syringe is pulled by the other hand to suck the used liquidculture medium in the vessel. The culture period of cells is typicallyabout two weeks, and a culture medium is required to be exchanged everyseveral days. Thus, the culture medium exchange operation that iscomplicated and requires a lot of skill must be carried out in eachcase. Such a manual culture medium exchange operation requires a lot oflabor and time. When many RWVs are run, the exchange operation becomes aheavy burden as well as unexpected environmental contamination may occurduring the culture medium exchange operation. In addition, the vesselhaving the cell suspension inlet, the supply port, and the dischargeport on the front face has an disadvantage of small view for theobservation of cells in the vessel.

To address this, the present applicant has developed an automatic cellculture apparatus as disclosed in Patent Document 4. Namely, theautomatic cell culture apparatus includes a closed housing and arotating culture device in the closed housing. The rotating culturedevice includes a cylindrical culture container having a horizontalrotating shaft, a cell suspension inlet, a supply port, and a dischargeport, and each of the supply port and the discharge port has a septumseal structure for liquid culture medium exchange. The supply port andthe discharge port are provided as a pair on the outer circumferentialcylindrical face of the culture container at positions 180° apart fromeach other with respect to the rotating center toward the radialdirection. The automatic cell culture apparatus also includes a syringeshifting means for inserting/withdrawing an injection needle of a supplysyringe containing a new liquid culture medium to/from the supply portand for inserting/withdrawing an injection needle of an empty dischargesyringe to/from the discharge port while locating the supply port andthe discharge port on a vertical line with the supply port facing upwardand includes a piston driving means for pushing a piston of the supplysyringe while inserting the supply syringe into the supply port and forsimultaneously pulling a piston of the discharge syringe while insertingthe discharge syringe into the discharge port.

The apparatus thereby has a function of automatically exchanging aliquid culture medium in the rotating culture device using the RWV.Hence, such an apparatus can largely reduce burdens of culture operatorssuch as researchers and achieve efficient cell culture as well asminimize the possibility of contamination in culture. When a pluralitypairs of the supply ports and the discharge ports are provided on theouter circumferential cylindrical face of the culture container at anequal angle interval depending on a culture period and the number ofliquid culture medium exchanges, the supply port and the discharge portcan be newly used for every liquid culture medium exchange. Thus, suchan apparatus has an advantage of minimizing the contamination during theliquid culture medium exchange.

Citation List

Patent Literature

Patent Document 1: JP-A No. 2006-014675

Patent Document 2: JP⁻A No. 2006-115798

Patent Document 3: JP-A No. 2006-149268

Patent Document 4: JP⁻A No. 2008-237203

SUMMARY OF INVENTION Technical Problem

However, the automatic cell culture apparatus disclosed in PatentDocument 4 still has some problems. That is, air may not be completelysubstituted at the initial charging of a cell suspension to remain inthe vessel, or air may be introduced into the vessel in culture througha permeable membrane on the backside. Accumulated gas in the vesseldisturbs the flow of a liquid culture medium to provide irregular loadto cells during rotation culturing, and hence air bleeding is essential.In conventional exchange of a liquid culture medium, a vessel ispositioned with the supply port and the discharge port for a liquidculture medium in the vertical direction and with the supply portlocating at the upper position, and a liquid culture medium is suppliedwith the supply syringe from the top and is sucked with the dischargesyringe from the bottom. Hence, gas accumulated in the upper part of thevessel cannot be bled.

Therefore, in view of the above circumstances, it is an object of thepresent invention to provide a rotating culture vessel that can automatecell seeding, liquid culture medium exchange, quality control, and thelike based on the rotation culture technique using the RWV and that cansimultaneously bleed air during the liquid culture medium exchangewithout disturbing cultured cells and an automatic cell cultureapparatus using the vessel.

Solution to Problem

In order to solve the above problems, the present invention provides arotating culture vessel used for three-dimensional culture of a cell ina nearly gravity-free microgravity environment, the rotating culturevessel including a cell and a liquid culture medium and being attachedto a horizontal rotating shaft of a rotating culture device. Therotating culture vessel is characterized by including one or moreinlets/outlets for supplying a cell and a liquid culture medium at ainitial stage and taking out a cultured cell at an appropriate positionon a flat cylindrical culture container, and at least one pair of asupply port and a discharge port for liquid culture medium exchange onan outer circumferential cylindrical face of the culture container. Thepair of the supply port and the discharge port are positioned 180°opposite to each other, the discharge port has a center line passingthrough a rotating center, and the supply port has a center lineeccentrically positioned with respect to the rotating center (claim 1).

It is more preferable that a distance between the center line of thesupply port and the rotating center is 0.5r to 0.9r where the culturecontainer has a culture space having a radius of r (claim 2).

It is also preferable that three pairs of the supply ports and thedischarge ports are provided on the outer circumferential cylindricalface of the culture container and the supply ports and the dischargeports are provided at an equal angle interval (claim 3).

It is preferable that each of the supply port and the discharge port hasa septum seal structure, a supply syringe used for liquid culture mediumexchange has a leading end having an injection needle optionally passingthrough the septum seal, a discharge syringe used for liquid culturemedium exchange has a leading end having an injection needle optionallypassing through the septum seal, a new liquid culture medium is suppliedfrom the supply syringe while a used liquid culture medium is suckedwith the discharge syringe, and air is optionally bled with thedischarge syringe (claim 4).

The outer circumferential cylindrical face of the culture containerincludes a cell suspension supply port for supplying a cell suspensioncomposed of a cell and a liquid culture medium, a front face orthogonalto the rotating shaft of the culture container includes an air bleedingport and a cell discharge port, each of the cell suspension supply portand the air bleeding port has a septum seal structure, and the celldischarge port has a rubber plug structure with a large opening (claim5).

Each port having the septum seal structure includes an inlet flow pathhaving a large diameter for accepting the injection needle behind theseptum seal and an orifice flow path having a small diameter between theinlet flow path and the culture space of the culture container (claim6).

The present invention also provides an automatic cell culture apparatususing the rotating culture vessel and used for three-dimensional cultureof a cell in a nearly gravity-free microgravity environment. Theautomatic cell culture apparatus is characterized by including a closedhousing having an air conditioning function, an incubator box of arotating culture device in a middle chamber of the closed housing, acool supply box storing a supply syringe for supplying a liquid culturemedium in a lower chamber of the closed housing, and a cool dischargebox storing a discharge syringe for collecting a liquid culture mediumin an upper chamber of the closed housing. Each of the incubator box,the cool supply box, and the cool discharge box includes a front facehaving an automatically operated door. The rotating culture device has ashaft direction shifting means shifting a horizontal rotating shaft backand forth in a shaft direction, the horizontal rotating shaft isprovided in the incubator box, and the rotating shaft has an endremovably installed with the rotating culture vessel. A front space ofthe cool supply box includes an XYZ-axis shifting mechanism for supply,the XYZ-axis shifting mechanism for supply includes a movable part forsupply driven by the mechanism, and the movable part for supply has afixing chuck for holding the supply syringe upward and a push up meansfor pushing a piston up. A front space of the cool discharge boxincludes an XYZ-axis shifting mechanism for discharge, the XYZ-axisshifting mechanism for discharge includes a movable part for dischargedriven by the mechanism, and the movable part for discharge has a fixingchuck for holding the discharge syringe downward and a pull up means forpulling a piston up. Each door is opened, the shaft direction shiftingmeans is driven to bring the rotating culture vessel from the incubatorbox to a front space, and the rotating culture vessel is stopped withthe supply port facing downward and the discharge port facing upward.The XYZ-axis shifting mechanism for supply and the fixing chuck aredriven to take out the supply syringe from the cool supply box and tolocate the supply syringe below the rotating culture vessel, and theXYZ-axis shifting mechanism for discharge and the fixing chuck aredriven to take out the discharge syringe from the cool discharge box andto locate the discharge syringe above the rotating culture vessel. TheXYZ-axis shifting mechanism for supply is driven to airtightly connectthe supply syringe to the supply port, simultaneously the XYZ-axisshifting mechanism for discharge is driven to airtightly connect thedischarge syringe to the discharge port, and then the push up means andthe pull up means are synchronously driven to exchange a liquid culturemedium in the rotating culture vessel (claim 7).

Each of the cool supply box and the cool discharge box includes arotating revolver type stacker and a plurality of holders around thestacker. The stacker has a vertical rotating shaft controlled with astepping motor, and each holder elastically removably holds the syringein a transverse direction with the syringe in the vertical direction(claim 8).

It is preferable that the rotating culture device includes a pluralityof the rotating shafts shifted back and forth by a common shaftdirection shifting means, the rotating shafts are not overlapped in thevertical direction, ends of the rotating shafts are displaced back andforth, and the adjacent rotating culture vessels do not interfere witheach other in the case of the rotating culture vessels being installedto the corresponding rotating shafts (claim 9).

Advantageous Effects of Invention

The rotating culture vessel of the present invention as described aboveincludes one or more inlets/outlets for supplying cells and a liquidculture medium at a initial stage and taking out cultured cells at anappropriate position on a flat cylindrical culture container and atleast one pair of a supply port and a discharge port for liquid culturemedium exchange on an outer circumferential cylindrical face of theculture container. The pair of the supply port and the discharge portare positioned 180° opposite to each other, the discharge port has acenter line passing through a rotating center, and the supply port has acenter line eccentrically positioned with respect to the rotatingcenter. Hence, the rotating culture vessel is stopped with the supplyport facing downward and the discharge port facing upward, the supplysyringe including a new liquid culture medium is connected to the supplyport, while the discharge syringe is connected to the discharge port,the piston of the supply syringe is pushed up to supply the new liquidculture medium into the culture container, and the piston of thedischarge syringe is simultaneously pulled up to suck a used liquidculture medium for liquid culture medium exchange, as well as at thetime, gas accumulated in the upper part of the culture container can besucked with the discharge syringe. The center line of the supply port iseccentrically positioned with respect to the rotating center, hence,even when clumps of cells in culture are settled down to be placed inthe lower part of the culture container, disassembling of the clumps ofcells due to the flow caused by the supply of the liquid culture mediumcan be suppressed because the supply port is placed avoiding the clumpsof cells. In particular, the effect is increased when the distancebetween the center line of the supply port and the rotating center is0.5r to 0.9r where the culture container has a culture space having aradius of r. The culture container does not have a supply port and adischarge port interrupting the view on the front face, and thus theprogress of culture is easily observed.

Three pairs of the supply ports and the discharge ports are provided onthe outer circumferential cylindrical face of the culture container, andthus a liquid culture medium can be exchanged three times. Hence, theculturing can be continued for about two weeks that is a common cellculture period, and the supply port and the discharge port can be newlyused for every exchange of a liquid culture medium. Therefore, theoccurrence of contamination can be minimized. When the supply ports andthe discharge ports are provided at an equal angle interval, the supplyport and the discharge port can be readily located at exact positions.

When each of the supply port and the discharge port has a septum sealstructure and each of the supply syringe and the discharge syringe usedfor liquid culture medium exchange has a leading end with an injectionneedle that can pass through the septum seal, the passing of theinjection needle through the septum seal can achieve airtightconnection, and a new liquid culture medium is supplied from the supplysyringe while a used liquid culture medium is sucked with the dischargesyringe as well as air can be bled with the discharge syringe.Furthermore, when the injection needle is removed from the septum seal,the hole formed with the injection needle is self-repaired to be closed.Thus, the supply port and the discharge port do not require a specialstructure for switching operation, and the supply port and the dischargeport can be downsized. Therefore, the outer circumferential cylindricalface of the culture container can have three pairs of the supply portsand the discharge ports.

The outer circumferential cylindrical face of the culture containerincludes the cell suspension supply port for supplying a cell suspensioncomposed of cells and a liquid culture medium, and a front faceorthogonal to the rotating shaft of the culture container includes anair bleeding port and a cell discharge port. Hence, the culturecontainer is kept in a horizontal position with the front face facingupward, a cell suspension supply syringe is connected to the cellsuspension supply port, an air discharge syringe is connected to the airbleeding port, a cell suspension is supplied from the cell suspensionsupply syringe, air in the culture container is simultaneously suckedwith the air discharge syringe, the air in the culture container isreplaced with the cell suspension, and initial culture preparation canbe completed. In particular, when each of the cell suspension supplyport and the air bleeding port has the septum seal structure, aninsertion of the injection needle of the syringe to the septum seal cansimply achieve airtight connection to lead to easy manual operation. Thecell discharge port has a rubber plug structure with a large opening,and hence clumps of cells can be readily taken out after culture.

Each port having the septum seal structure includes the inlet flow pathhaving a large diameter for accepting the injection needle behind theseptum seal and the orifice flow path having a small diameter betweenthe inlet flow path and the culture space of the culture container.Thus, even when the injection needle is inserted into the septum seal ata slightly dislocated position, the injection needle can be acceptedinto the inlet flow path, and the supply syringe can supply a liquidculture medium through the orifice flow path into the culture space.Hence, such a structure can minimize the disturbance in the culturespace due to the liquid culture medium flow and can suppress theintroduction of air remaining in the inlet flow path through the orificeflow path into the culture space in rotation culture. Furthermore, thecircumferential face of the culture space has a small hole of theorifice flow path alone, and thus the liquid culture medium is notdisturbed in rotation culture.

The automatic cell culture apparatus of the present invention is basedon the rotation culture technique using RWVs and has the function ofautomatic liquid culture medium exchange. Thus, such an apparatus canlargely reduce burdens of culture operators such as researchers toachieve efficient cell culture as well as can minimize the possibilityof contamination in culture. Hence, even medical institutions not havingCPC in compliance with GMP can expect clinical application ofregenerative medicine, and consequently, the regenerative medicine canbe greatly generalized. The present invention can completely automatethe liquid culture medium exchange operation that has been proven byconventional manual operation in which a new liquid culture medium issupplied into a culture container in a rotating culture vessel from asupply port with a supply syringe and a used liquid culture medium issimultaneously sucked from a discharge port with a discharge syringe.That is, the new liquid culture medium is supplied into the culturespace of the rotating culture vessel from the supply port facingdownward with the supply syringe, the used liquid culture medium issimultaneously sucked from the discharge port facing upward with thedischarge syringe, and concurrently air can be bled. The center line ofthe supply port is eccentrically positioned with respect to the rotatingcenter, and thus the disassembling of the clumps of cells can besuppressed as described above. The used liquid culture medium sucked inthe discharge syringe is stored in the cool discharge box, and hence thecontamination can be checked later.

Typically, each of the supply port and the discharge port is used onlyonce for the liquid culture medium exchange, but the rotating culturevessel has a comparatively small culture space, for example, a smallvessel has a volume of 10 ml and a large vessel has a volume of 20 ml.Thus, the numbers of the supply ports and the discharge ports arelimited on the outer circumference. In the case of a long termculturing, each of the supply port and the discharge port may be usedtwice or more. In such a case, the numbers of the supply syringes andthe discharge syringes require more than the number of the supply ports,but the use of a rotating revolver type stacker having a plurality ofholders around the stacker can achieve the supply and the storage of thesupply syringes and the discharge syringes.

Even in the case that each of the supply port and the discharge port isused once for the liquid culture medium exchange, when the rotatingculture device includes a plurality of rotating shafts, a plurality ofrotating culture vessels are installed to the ends, and a plurality ofcell lines are simultaneously cultured, it is required that the numbersof the supply syringes and the discharge syringes, obtained bymultiplying the number of the vessels by the number of pairs of thesupply ports and the discharge ports. In such a case, the rotatingrevolver type stacker having a plurality of holders around the stackercan be used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a general perspective view of an automatic cell cultureapparatus of the present invention except for a part of an outer cover.

FIG. 2 is a side view showing the internal structure of the automaticcell culture apparatus of the present invention.

FIG. 3 is an elevation view showing the internal structure of theautomatic cell culture apparatus of the present invention.

FIG. 4 is a perspective view showing a mechanism part of a rotatingculture device having a shaft direction shifting means.

FIG. 5 is a perspective view showing a XYZ-axis shifting mechanism forsupply.

FIG. 6 is a perspective view showing a XYZ-axis shifting mechanism fordischarge.

FIG. 7 is a perspective view of a cool supply box having a rotatingrevolver type stacker.

FIG. 8 is a perspective view of a cool discharge box having the rotatingrevolver type stacker.

FIG. 9 is a perspective view of a rotating culture vessel of the presentinvention viewed from the front side.

FIG. 10 is a perspective view of the rotating culture vessel of thepresent invention viewed from the back side.

FIG. 11 is an elevation view of the rotating culture vessel.

FIG. 12 is a left side view of the rotating culture vessel.

FIG. 13 is a back view of the rotating culture vessel.

FIG. 14 is a cross-sectional view taken along the line A-A in FIG. 11.

FIG. 15 is a cross-sectional view taken along the line B-B in FIG. 11.

FIG. 16 is a schematic diagram showing the liquid culture mediumexchange operation using the first pair of the supply port and thedischarge port.

FIG. 17 is a schematic diagram showing the liquid culture mediumexchange operation using the second pair of the supply port and thedischarge port.

FIG. 18 is a schematic diagram showing the liquid culture mediumexchange operation using the third pair of the supply port and thedischarge port.

FIG. 19 is a photograph in place of a drawing, showing externalappearances of cartilage tissues as the results of cartilage tissueformation experiment using bone marrow cells of Japanese white rabbitsusing the automatic cell culture apparatus of the present invention(automatic culture) and a conventional rotating culture device (manualculture).

FIG. 20 is a graph showing the result comparing the production amountsof GAG in cartilage matrix.

FIGS. 21 are micrographs of cartilage tissues stained with Alcian blue.

FIGS. 22 are micrographs of cartilage tissues stained with toluidineblue.

FIGS. 23 are micrographs of cartilage tissues stained with HE.

DESCRIPTION OF EMBODIMENTS

Next, the present invention will be described in further detail based onembodiments shown in attached drawings. FIG. 1 to FIG. 8 show theautomatic cell culture apparatus of the present invention, and FIG. 9 toFIG. 15 show the rotating culture vessel of the present invention. InFigures, the sign 1 represents the automatic cell culture apparatus, thesign 2 represents the rotating culture vessel, the sign 3 represents arotating culture device, the sign 4 represents a cool supply box, thesign 5 represents a cool discharge box, the sign 6 represents a shaftdirection shifting means, the sign 7 represents an XYZ-axis shiftingmechanism for supply, and the sign 8 represents an XYZ-axis shiftingmechanism for discharge.

The automatic cell culture apparatus 1 of the present invention uses arotating culture vessel 2 and is used for three-dimensional culture ofcells in a microgravity environment that is nearly gravity-free. Theautomatic cell culture apparatus includes a closed housing 9 having anair conditioning function, the closed housing 9 includes a middlechamber that includes a rotating culture device 3 having an incubatorbox 10 in which one or more rotating culture vessels 2 are installed forculture, a lower chamber that includes a cool supply box 4 storing asupply syringe 11 for supplying a liquid culture medium, and an upperchamber that includes a cool discharge box 5 storing a discharge syringe12 for collecting a liquid culture medium. Each of the incubator box 10,the cool supply box 4, and the cool discharge box 5 includes a frontface having an automatically operated door (not shown in the drawings).The rotating culture device 3 has a shaft direction shifting means 6shifting a horizontal rotating shaft 13 provided in the incubator box 10back and forth in the shaft direction, and the rotating shaft 13 has anend that is removably installed with the rotating culture vessel 2. Afront space of the cool supply box 4 includes an XYZ-axis shiftingmechanism 7 for supply, the XYZ-axis shifting mechanism 7 for supplyincludes a movable part for supply driven by the mechanism, and themovable part for supply has a fixing chuck 14 for holding the supplysyringe 11 upward and a push up means 15 for pushing a piston 11A up. Afront space of the cool discharge box 5 includes an XYZ-axis shiftingmechanism 8 for discharge, the XYZ-axis shifting mechanism 8 fordischarge includes a movable part for discharge driven by the mechanism,and the movable part for discharge has a fixing chuck 16 for holding thedischarge syringe 12 downward and a pull up means 17 for pulling apiston 12A up. In the present embodiment, the shaft direction of therotating shaft 13 is regarded as an X axis, the horizontal directionorthogonal to the X axis is regarded as a Y axis, and the verticaldirection is regarded as a Z axis for description.

In the present embodiment, a plurality of rotating shafts 13 that areshifted back and forth in the X axis direction by a common shaftdirection shifting means 6 are provided while the rotating shafts arenot overlapped in the vertical direction. Ends of the rotating shafts 13are displaced back and forth so that the adjacent rotating culturevessels 2 will not interfere with each other when the rotating culturevessels 2 are installed to the corresponding rotating shafts 13. In thepresent embodiment, two rotating shafts 13 are provided in parallel toachieve cell culture using two rotating culture vessels 2 at the sametime.

Next, the rotating culture vessel 2 will be described in detail based onFIG. 9 to FIG. 15. The rotating culture vessel 2 includes a cellsuspension supply port 19 for supplying a cell suspension composed ofcells and a liquid culture medium on an outer circumferentialcylindrical face of a flat cylindrical culture container 18 as well asincludes an air bleeding port 20 and a cell discharge port 21 on a frontface orthogonal to the rotating shaft 13 of the culture container 18.The rotating culture vessel 2 further includes at least one pair of asupply port 22 and a discharge port 23 for liquid culture mediumexchange on the outer circumferential cylindrical face of the culturecontainer 18. The pair of the supply port 22 and the discharge port 23is positioned 180° opposite to each other, the discharge port 23 has acenter line passing through the rotating center, and the supply port 22has a center line eccentrically positioned with respect to the rotatingcenter.

In the present embodiment, three pairs of the supply ports 22 and thedischarge ports 23 are provided on the outer circumferential cylindricalface of the culture container 18, and these ports will be distinguishedby A, B, and C. The supply ports 22A, 22B, and 22C are provided at anequal angle interval and the discharge ports 23A, 23B, and 23C are alsoprovided at an equal angle interval. In the present embodiment, the cellsuspension supply port 19 and the cell discharge port 21 areindependently provided, but one port may be provided to serve as bothports. Here, the distance between the center line of the supply port 22and the rotating center is 0.5r to 0.9r where the culture container 18has a culture space having a radius of r.

The rotating culture vessel 2 further includes, at the back center, aprotruding attaching part 24 that is removably installed to the end ofthe rotating shaft 13 in the rotating culture device 3. The attachingpart 24 has a structure so that the vessel will be attached at the samerotational position with respect to the end of the rotating shaft 13 atany time. The rotating culture vessel 2 of the embodiment is expected tohave a volume of 10 to 20 ml because a liquid culture medium isexpensive, but the volume should be determined depending on the size ofclumps of cells to be cultured.

As shown in FIG. 10, FIG. 13, FIG. 14, and FIG. 15, the rotating culturevessel 2 has air intake ports 25 on the back side around the attachingpart 24, and a gas permeable membrane 26 is provided inside the airintake port 25. Through the membrane, oxygen can be supplied into aliquid culture medium while carbon dioxide can be discharged. Therotating culture vessel 2 also has an observation window 27 on the frontside so that the inside can be observed.

As shown in FIG. 15, each of the supply port 22 and the discharge port23 has a septum seal structure. The supply syringe 11 and the dischargesyringe 12 used for liquid culture medium exchange have injectionneedles 11B and 12B, respectively, at the leading ends, and eachinjection needle can pass through the septum seal 28. The supply port 22is positioned downward, and the discharge port 23 is positioned upward.A new liquid culture medium is supplied from the lower side with thesupply syringe 11 while a used liquid culture medium is sucked from theupper side with the discharge syringe 12 and air can be bled with thedischarge syringe 12. Each of the supply port 22 and the discharge port23 has the septum seal structure, and thus the injection needles 11B and12B installed to the leading ends of the supply syringe 11 and thedischarge syringe 12 pass through the septum seals 28 to achieveairtight connection. Moreover, even when the injection needles 11B and12B are removed, the through holes are closed due to elastic recovery ofthe septum seal to maintain the airtight condition.

More specifically, the supply port 22 and the discharge port 23 have thesame cross sectional structure as shown in FIG. 15. The supply port 22includes a port 29 that is eccentrically positioned with respect to therotating center of the outer circumferential cylindrical face of theculture container 18 and continues to the culture space. The supply port22 further includes a silicon rubber septum seal 28 in an end part ofthe port 29. The circumference of the septum seal 28 is pressed with apressing cap 30 screwed with the port 29 to lead to a closed structure.Meanwhile, the discharge port 23 includes a port 29 that continues tothe culture space toward the radial direction passing through therotating center of the outer circumferential cylindrical face of theculture container 18. The discharge port 23 further includes a siliconrubber septum seal 28 in an end part of the port 29. The circumferenceof the septum seal 28 is pressed with a pressing cap 30 screwed with theport 29 to lead to a closed structure. Each port 29 constituting thesupply port 22 and the discharge port 23 having the septum sealstructures includes an inlet flow path 31 having a large diameter behindthe septum seal 28 and an orifice flow path 32 having a small diameterbetween the inlet flow path 31 and the culture space in the culturecontainer 18, and the inlet flow path 31 accepts the injection needle11B or 12B.

The leading end of the injection needle 11B of the supply syringe 11passed thorough the septum seal 28 of the supply port 22 is accepted inthe inlet flow path 31 in the port 29, and thus even when the injectionneedle 11B is inserted into the septum seal 28 slightly dislocated fromthe center, the leading end of the injection needle 11B does not come incontact with the port 29 to be used without problems. Hence, request tothe positioning precision of the XYZ-axis shifting mechanism 7 forsupply is lowered, and the mechanism can be formed using a cheapactuator. The orifice flow path 32 is also provided. Thus, a liquidculture medium supplied in the inlet flow path 31 is introduced throughthe orifice flow path 32 having a small diameter into the culture space,and the disturbance generated in the culture space can be minimized. Theleading end of the injection needle 12B of the discharge syringe 12passed through the septum seal 28 of the discharge port 23 is alsoaccepted in the inlet flow path 31 in the port 29, and a used liquidculture medium and air accumulated in the culture space can bedischarged through the orifice flow path 32 by suction. Even when air isaccumulated in the inlet flow path 31, the air does not flow into theculture space through the orifice flow path 32 in rotation culture dueto the surface tension of an liquid culture medium.

As shown in FIG. 14, each of the cell suspension supply port 19 and theair bleeding port 20 has a septum seal structure similar to the above,and the cell discharge port 21 has a rubber plug structure having alarge opening. That is, as with the discharge port 23, the cellsuspension supply port 19 includes a port 29 that continues to theculture space toward the radial direction passing through the rotatingcenter. The cell suspension supply port 19 also includes a siliconrubber septum seal 28 in an end part of the port 29. The circumferenceof the septum seal 28 is pressed with a pressing cap 30 screwed with theport 29 to lead to a closed structure. The air bleeding port 20 and thecell discharge port 21 are positioned on the periphery of the front faceof the culture container 18 opposite to each other with respect to therotating center. The air bleeding port 20 includes a port 33 protrudedfrom the front face of the culture container 18 and a silicon rubberseptum seal 34 in an end part of the port 33. The circumference of theseptum seal 34 is pressed with a pressing cap 35 screwed with the port33 to lead to a closed structure. Each of the port 29 of the cellsuspension supply port 19 and the port 33 of the air bleeding port 20includes an inlet flow path 31 and an orifice flow path 32 in a similarmanner to the above. An inside where the orifice flow path 32 of the airbleeding port 20 is opened toward the culture space has a concave part36 in which air in the culture space is accumulated when the rotatingculture vessel 2 is horizontalized. The cell discharge port 21 includesa port 37 having a large inner diameter, and the port is tightly fittedwith a rubber plug 38. The rubber plug 38 has a leading end face that isflush with the inner wall of the culture space so as not to disturb aliquid culture medium in rotation culture.

In order to supply cells and a liquid culture medium into the rotatingculture vessel 2, the rotating culture vessel 2 is left with theobservation window 27 facing upward and the rotating shaft in thevertical direction, an injection needle of a cell suspension supplysyringe (not shown in the drawings) containing the cells and the liquidculture medium is inserted into the septum seal 28 of the cellsuspension supply port 19, an injection needle of an empty air bleedingsyringe (not shown in the drawings) is inserted into the septum seal 34of the air bleeding port 20, and the cell suspension is supplied fromthe cell suspension supply syringe into the culture space while air isbled with the air bleeding syringe. At the time, the air in the culturespace is finally accumulated in the concave part 36, and the airaccumulated in the concave part 36 is discharged from the culture space.

Next, the automatic cell culture apparatus 1 for culturing cells whileautomatically exchanging a liquid culture medium using the rotatingculture vessel 2 will be described in detail based on FIG. 1 to FIG. 8and FIG. 16 to FIG. 18. As shown in FIG. 1, the closed housing 9 hasopening and closing doors on the front face and one side face. The frontface has three inspection doors 39 corresponding to the rotating culturedevice 3, the cool supply box 4, and the cool discharge box 5. Theclosed housing 9 has a transparent front panel so that the inside can beobserved. The closed housing 9 includes the side face having oneoperation door 40 for the preparation of a series of culture or for thetreatment after culture. That is, the operation door 40 is opened, therotating culture vessel 2 is attached to or removed from the rotatingshaft 13, the supply syringes 11 are attached to or removed from theinside of the cool supply box 4, and the discharge syringes 12 areattached to or removed from the inside of the cool discharge box 5. Theoperation door 40 is transparent so that the inside can be observed. Theclosed housing 9 includes a ceiling having an air conditioner 41 withfilter function for keeping the inside at suitable temperature and forair cleaning.

As shown in FIG. 1 to FIG. 4, the rotating culture device 3 includes theincubator box 10 in which the temperature can be controlled and thatcontains a horizontal rotating shaft 13 as well as includes a rotationcontrol mechanism 42 behind the incubator box, for driving the rotatingshaft 13 at a predetermined rotation speed, and a shaft directionshifting means 6 for shifting the rotating shaft 13 together with therotation control mechanism 42 back and forth in the shaft direction. Theincubator box 10 has an automatically operated door on the front faceand a manually operated door 43 on the side face having the operationdoor 40. In the present embodiment, two rotating shafts 13 are arrangedin parallel in a horizontal position, and each rotating shaft issupported with a cylindrical shaft bearing 44 rotatably and slidably inthe shaft direction. The shaft direction shifting means 6 has astructure to shift a movable part 46 in front and back directions alonga linear guide 45 provided behind the incubator box 10. Ends of therotating shafts 13 continuing backward from the back face of theincubator box 10 are interlocked by a rotation control mechanism 42 thatis attached to the movable part 46 of the shaft direction shifting means6 through a timing belt. There are two independent rotation controlmechanisms 42 so as to independently control each rotation of therotating shafts 13. The rotation control mechanism 42 is driven by astepping motor or a servomotor so as to accurately control the rotationspeed and the rotational position and controls the rotational positionby, for example, reading a marker fixed on the rotating shaft 13 with asensor. The leading end positions of the rotating shafts 13 aredisplaced back and forth as described above, and the rotating culturevessel 2 filled with a cell suspension is manually attached to orremoved from each leading end.

As shown in FIG. 1 to FIG. 3 and FIG. 7, the cool supply box 4 includesan automatically operated door on the front face of the box in whichtemperature can be controlled, a manually operated door 47 on the sideface having the operation door 40, and a rotating revolver type stacker48 in the cool supply box. The stacker 48 includes a vertical rotatingshaft 50 that is controlled with a stepping motor 49 and a plurality ofholders 51 around the stacker 48. The holder 51 elastically removablyholds the supply syringe 11 in a transverse direction with the injectionneedle 11B facing upward in the vertical direction. The holder 51 of thestacker 48 include an U-groove that accepts and locks the upper andlower parts of the supply syringe 11 and a grip that elastically gripsthe supply syringe 11 from both sides for holding. The supply syringe 11is laterally pushed to be automatically held with the holder, and thesupply syringe 11 is grasped and laterally pulled from the holder to bereadily removed.

As shown in FIG. 1 to FIG. 3 and FIG. 8, the cool discharge box 5 hasapproximately the same structure as that of the cool supply box 4, andincludes an automatically operated door on the front face of the box inwhich temperature can be controlled, a manually operated door 52 on theside face having the operation door 40, and a rotating revolver typestacker 53 in the cool discharge box 5. The stacker 53 includes avertical rotating shaft 55 that is controlled with a stepping motor 54and a plurality of holders 56 around the stacker 53. The holderelastically removably holds the discharge syringe 12 in a transversedirection with the injection needle 12B facing downward in the verticaldirection. The stacker 53 has approximately the same structure as thatof the stacker 48.

Next, the XYZ-axis shifting mechanism 7 for supply for transferring thesupply syringe 11 will be described based on FIG. 2, FIG. 3, and FIG. 5.The XYZ-axis shifting mechanism 7 for supply is provided in a frontspace of the cool supply box 4 in the closed housing 9. The XYZ-axisshifting mechanism 7 for supply includes a Y-axis shifting mechanism 57fixed to the bottom in the closed housing 9, a Z-axis shifting mechanism58 fixed to a movable part of the Y-axis shifting mechanism 57, and anX-axis shifting mechanism 59 fixed to a movable part of the Z-axisshifting mechanism 58. The X-axis shifting mechanism 59 includes amovable part having a fixing chuck 14 for holding the supply syringe 11upward and having a push up means 15 for pushing a piston 11A up. Eachof the Y-axis shifting mechanism 57, the Z-axis shifting mechanism 58,and the X-axis shifting mechanism 59 is composed of a linear guide and astepping motor driven with a ball screw, but the structure is notspecifically limited. The fixing chuck 14 is composed of a U-grooveplate 60 that locks a flange part of a cylinder of the supply syringe 11and an air-driven hand 61 that grips the side face of the cylinder. Thepush up means 15 includes a protruded push up plate 63 that is providedon a movable part of a Z-axis shifting mechanism 62 and that is incontact with the lower end of the piston 11A. The movable part of theZ-axis shifting mechanism 62 along with the fixing chuck 14 are fixed tothe movable part of the X-axis shifting mechanism 59.

Finally, the XYZ-axis shifting mechanism 8 for discharge fortransferring the discharge syringe 12 will be described based on FIG. 2,FIG. 3, and FIG. 6. The XYZ-axis shifting mechanism 8 for discharge isprovided in a front space of the cool discharge box 5 in the closedhousing 9. The XYZ-axis shifting mechanism 8 for discharge includes aY-axis shifting mechanism 64 fixed to an anterior top area in the closedhousing 9, a Z-axis shifting mechanism 65 fixed to a movable part of theY-axis shifting mechanism 64, and an X-axis shifting mechanism 66 fixedto a movable part of the Z-axis shifting mechanism 65. The X-axisshifting mechanism 66 includes a movable part having a fixing chuck 16for holding the discharge syringe 12 downward and a pull up means 17 forpulling a piston 12A up. The fixing chuck 16 is composed of a U-grooveplate 67 that locks a flange part of a cylinder of the discharge syringe12 and an air-driven hand 68 that grips the side face of the cylinder.The pull up means 17 include a protruded pull up plate 70 that isprovided on a movable part of a Z-axis shifting mechanism 69 and thatlocks a flange part at the upper end of the piston 12A for pulling up.The movable part of the Z-axis shifting mechanism 69 along with thefixing chuck 16 are fixed to the movable part of the X-axis shiftingmechanism 66.

The procedure for culturing cells using the automatic cell cultureapparatus 1 of the present invention will be described below. First, theoperation door 40 of the closed housing 9 is opened, the door 47 of thecool supply box 4 is opened, a predetermined number of supply syringes11 are installed to the stacker 48, and the door 47 is closed. The door52 of the cool discharge box 5 is opened, an equal number of thedischarge syringes 12 to that of the supply syringes 11 are installed tothe stacker 53, and the door 52 is closed. Meanwhile, the door 43 of theincubator box 10 of the rotating culture device 3 is opened, theattaching part 24 of the rotating culture vessel 2 filled with a cellsuspension is installed to the end of the rotating shaft 13, and thedoor 43 is closed. At the time, the rotating culture vessel 2 is fixedto a precise rotational position with respect to the rotating shaft 13at any time. Next, the operation door 40 is closed, and the temperaturein the closed housing 9 together with the incubator box 10 is kept at apredetermined temperature. This prevents that the temperature in theincubator box 10 is steeply changed to change a culture condition whenthe automatically operated door on the front face of the incubator box10 is opened for liquid culture medium exchange. Each temperature in thecool supply box 4 and the cool discharge box 5 is controlled to be lowerthan the temperature in the incubator box 10 in order to store a newliquid culture medium before use and a used liquid culture mediumwithout varying the condition.

Then, the rotation control mechanism 42 is driven to rotate the rotatingculture vessel 2 at a predetermined rotation speed for cell culture.After culturing for a predetermined period of time, the rotation controlmechanism 42 is controlled to stop the rotating culture vessel 2 withthe first supply port 22A facing downward in the vertical direction andthe first discharge port 23A facing upward in the vertical direction.Next, the automatically operated door on the front face of the incubatorbox 10 is opened, the shaft direction shifting means 6 is driven toshift the rotating culture vessel 2 forward, and the rotating culturevessel 2 is located in a front space of the incubator box 10.Simultaneously or before or after that, each automatically operated dooron the front faces of the cool supply box 4 and the cool discharge box 5is opened, each of the XYZ-axis shifting mechanism 7 for supply and theXYZ-axis shifting mechanism 8 for discharge is independently driven, thefixing chuck 14 is transferred into the cool supply box 4, the supplysyringe 11 stored in the stacker 48 at a predetermined rotationalposition is held with the fixing chuck 14, then the supply syringe 11 istransferred into a front space of the cool supply box 4, as well as thefixing chuck 16 is transferred into the cool discharge box 5, thedischarge syringe 12 stored in the stacker 53 at a predeterminedrotational position is held with the fixing chuck 16, and then thedischarge syringe 12 is transferred into a front space of the cooldischarge box 5.

Next, the supply syringe 11 is located directly below the first supplyport 22A of the rotating culture vessel 2, while the discharge syringe12 is located directly above the discharge port 23A. Then, as shown inFIG. 16, the Z-axis shifting mechanism 58 of the XYZ-axis shiftingmechanism 7 for supply and the Z-axis shifting mechanism 65 of theXYZ-axis shifting mechanism 8 for discharge are synchronously driven,the injection needle 11B of the supply syringe 11 is passed through theseptum seal 28 of the supply port 22A for connection, andsimultaneously, the injection needle 12B of the discharge syringe 12 ispassed through the septum seal 28 of the discharge port 23A forconnection. While maintaining this condition, the push up plate 63 ofthe push up means 15 is elevated to push the piston 11A of the supplysyringe 11 up, a new liquid culture medium is supplied into the culturespace of the rotating culture vessel 2, simultaneously, the pull upplate 70 of the pull up means 17 is elevated to pull the piston 12A ofthe discharge syringe 12 up, a used liquid culture medium is sucked outfrom the culture space of rotating culture vessel 2, and at the time,air accumulated in the culture space during culturing is alsosimultaneously sucked out. After the liquid culture medium in therotating culture vessel 2 is exchanged in this manner, the Z-axisshifting mechanism 58 of the XYZ-axis shifting mechanism 7 for supplyand the Z-axis shifting mechanism 65 of the XYZ-axis shifting mechanism8 for discharge are synchronously driven, the injection needle 11B ofthe supply syringe 11 is removed from the supply port 22A, andsimultaneously, the injection needle 12B of the discharge syringe 12 isremoved from the discharge port 23A. Next, the XYZ-axis shiftingmechanism 7 for supply and the XYZ-axis shifting mechanism 8 fordischarge are driven, the used supply syringe 11 and the used dischargesyringe 12 are installed to the original positions in the stacker 48 andthe stacker 53, respectively, the fixing chuck 14 and the fixing chuck16 are unlocked and returned from the cool supply box 4 and the cooldischarge box 5 to stay at the initial positions. Meanwhile, the shaftdirection shifting means 6 is driven to store the rotating culturevessel 2 in the incubator box 10, each automatically operated door isclosed, and then the rotation control mechanism 42 is driven to rotatethe rotating culture vessel 2 at a predetermined rotation speed for cellculture.

The liquid culture medium exchange operation is performed using a pairof the supply port 22B and the discharge port 23B of the rotatingculture vessel 2 as shown in FIG. 17, and then the operation is repeatedusing a pair of the supply port 22C and the discharge port 23C as shownin FIG. 18. In this case, each of the stacker 48 and the stacker 53 isrotated by a predetermined angle so that a new supply syringe 11 and anew discharge syringe 12 will be located at the front in sequence.

The rotating culture device 3 employed in the present invention can keepcells in suspension without settling in the rotating culture vessel 2.Therefore, the rotating culture device 3 has advantages thatthree-dimensional aggregates can be formed, necrosis due to stirringstress can be avoided, differentiation inducers effectively work, andremoval of waste products and supply of nutrients can be performed. Inthe present invention, a liquid culture medium can be sequentiallysupplied using a plurality of the supply syringes 11. Hence, a liquidculture medium having an optimum composition may be used depending on aculture stage of cells.

It is important to observe a culture condition in culture. Theobservation of the culture condition is carried out on, for example, (1)pH change and color change of a culture medium due to the consumption ofculture medium additives, accumulation of waste products, and the likein culture, (2) presence or absence of turbidity of a culture medium dueto contamination, and (3) whether a three-dimensional tissue is formedfrom floating cells. In the present invention, the rotating culturevessel 2 has the observation window 27 on the front face. Thus, theinner condition can be observed through an imaging camera or variousanalytical equipments placed toward the observation window 27, and anactual condition can be analyzed through image processing. Based on thecondition, the rotation control mechanism 42 can be feedback-controlled,and the timing of liquid culture medium exchange can be automaticallydetermined.

EXAMPLES

Next, rotation cultures were performed using the automatic cell cultureapparatus of the present invention and by hand as a control (usingRCCS-4D manufactured by Synthecon, the rotation speed was visuallycontrolled). Cartilage tissue formation experiment was carried out usingbone marrow cells of Japanese white rabbit to compare the both rotationcultures. The experimental procedure will be described below.

(Experimental Procedure)

(1) Bone marrow cells were collected from the long bones of two Japanesewhite rabbits, 10 days old, and suspended in 20 ml standard medium.

*Standard Medium: DMEM (Dulbeccco's modified Eagle's medium (DMEM,Sigma, St. Louis Mo.)+10% FBS (fetal bovineserum)+antibiotic-antimycotic (Invitrogen, Carlsbad, Calif.)

(2) Next, the cells were seeded into a 75T flask (BD) together with 15ml standard medium and cultured in 5% CO2 at 37° C. for 3 weeks.

(3) Next, the cells were removed with trypsin, suspended in a bioreactormedium, and transferred into a 50 cc vessel.

*Bioreactor Medium: DMEM+50 μg/ml ascorbic acid (WAKO)+40μg/m1L-proline+ITS culture supplement (BD Biosciences), 10-7dexamethasone (Sigma), 10 ng/ml TGF-β3 (Sigma), andabtibiotic-antimycotic (BD)

*The 50 cc vessel used in Example is that shown in FIG. 9 to FIG. 15.

*The number of cells used for the culture using the automatic cellculture apparatus (Example) was the same as that for the manual culture(Comparative Example).

(4) The cells were cultured for 2 weeks. The tissue was taken out,macroscopically observed, and sliced into sections. The sections wereevaluated by histochemical techniques.

During the liquid culture medium exchange by the automatic cell cultureapparatus of the present invention, cellular tissues were not hit by thevessel wall. Furthermore, the liquid culture medium did not leak betweeneach injection needle of the supply syringe and the discharge syringeand the septum seal. It could be observed that the used liquid culturemedium in the vessel was exchanged with the new liquid culture medium insequence from the bottom. There was no accumulated gas in the upper partof the vessel after the liquid culture medium exchange.

(Culture Result)

FIG. 19 shows the appearances of the cartilage tissues formed by thecultures. In FIG. 19, the left image shows the result of the automaticculture (Example) and the right image shows the result of the manualculture (Comparative Example). Macroscopic observations of the culturedcartilage tissues revealed that the tissue by the manual culture waslarger than that by the automatic culture.

FIG. 20 is a graph showing the result comparing the production amountsof GAG in the cartilage matrix. In this culture, the production amountby the automatic culture was higher. In the experiments repeated severaltimes, the production amount of GAG in the cartilage matrix by theautomatic culture was the same as or higher than that by the manualculture.

Finally, FIGS. 21 to FIGS. 23 show the results of the histologicalevaluations on the cartilage tissues formed by the cultures. FIGS. 21are micrographs showing the results of the cartilage tissues stainedwith Alcian blue. In both tissues, the cartilage matrix was stainedlight blue, and the rich production of the cartilage matrix wasconfirmed. Here, the area where the cartilage matrix was stained lightblue is indicated by dark color in FIGS. 21.

FIGS. 22 are micrographs showing the results of the cartilage tissuesstained with toluidine blue. In both tissues, the cartilage matrix wasstained blue purple, and the rich production of the cartilage matrix wasconfirmed. In this case, the area where the cartilage matrix was stainedblue purple is also indicated by dark color in FIGS. 22.

FIGS. 23 are micrographs showing the results of the cartilage tissuesstained with HE (hematoxylin-eosin). The cartilage tissue was stainedblue purple with hematoxylin, and the mature chondrocytes stained bluepurple were observed in both tissues by the automatic culture and themanual culture. In this case, the area stained blue purple is alsoindicated by dark color in FIGS. 23. However, cytoplasm, connectivetissue in soft tissue, red blood cells, fibrin, endocrine granules, andthe like are stained light red or indigo blue with eosin. Thus, eachstained area is indicated by similar dark color in a monochrome image,and the cartilage tissue cannot be distinguished in the monochromeimage.

As described above, the culture using the automatic cell cultureapparatus of the present invention was compared with that by hand whilecontrolling the rotation. The result of the culture of rabbit bonemarrow cells with the rotating culture device using the RWV vesselrevealed that the automatic culture was equal or superior in quality tothe manual culture.

INDUSTRIAL APPLICABILITY

According to the automatic cell culture apparatus of the presentinvention, even medical institutions not having cell processing center(CPC) in compliance with good manufacturing practice (GMP) can expectclinical application of regenerative medicine, and consequently, theregenerative medicine can be greatly generalized. Typically, it can beused in order to form transplantable cartilage tissues from human bonemarrow cells. In addition to the cartilage regeneration, the study ofthe regenerative medicine has been extended to corneal regeneration forretinal detachment, cataract, and the like, bone regeneration for bonedefect and osteoporosis, pancreas (Langerhans island) regeneration fordiabetes mellitus and the like, cardiac muscle regeneration for dilatedcardiomyopathy and the like, nerve regeneration for Parkinson's diseaseand Alzheimer's disease, and the like. Hence, the automatic cell cultureapparatus of the present invention is supposed to have an advantage inthe regenerative medicine in addition to the cartilage regeneration. Theautomatic cell culture apparatus of the present invention will begenerally applicable to the regenerative medicine in addition to thecartilage regenerative medicine in future, and will certainly be theessential and important basic technique for the generalization of theregenerative medicine.

Reference Signs List

1 Automatic cell culture apparatus

2 Rotating culture vessel

3 Rotating culture device

4 Cool supply box

5 Cool discharge box

6 Shaft direction shifting means

7 XYZ-axis shifting mechanism for supply

8 XYZ-axis shifting mechanism for discharge

9 Closed housing

10 Incubator box

11 Supply syringe

11A Piston

11B Injection needle

12 Discharge syringe

12A Piston

12B Injection needle

13 Rotating shaft

14 Fixing chuck

15 Push up means

16 Fixing chuck

17 Pull up means

18 Culture container

19 Cell suspension supply port °Air bleeding port <Cell discharge port

22, 22A, 22B, 22C Supply port

23, 23A, 23B, 23C Discharge port

24 Attaching part

25 Intake port

26 Gas permeable membrane

27 Observation window

28 Septum seal

29 Port

30 Cap

31 Inlet flow path

32 Orifice flow path

33 Port

34 Septum seal

35 Cap

36 Concave part

37 Port

38 Rubber plug

39 Inspection door

40 Operation door ″Air conditioner

42 Rotation control mechanism

43 Door

44 Shaft bearing

45 Linear guide

46 Movable part

47 Door

48 Stacker

49 Stepping motor

50 Rotating shaft

51 Holder

52 Door

53 Stacker

54 Stepping motor

55 Rotating shaft

56 Holder

57 Y-axis shifting mechanism

58 Z-axis shifting mechanism

59 X-axis shifting mechanism

60 U-groove plate

61 Hand

62 Z-axis shifting mechanism

63 Push up plate

64 Y-axis shifting mechanism

65 Z-axis shifting mechanism

66 X-axis shifting mechanism

67 U-groove plate

68 Hand

69 Z-axis shifting mechanism

70 Pull up plate

1. A rotating culture vessel used for three-dimensional culture of acell in a nearly gravity-free microgravity environment, the rotatingculture vessel including a cell and a liquid culture medium and beingattached to a horizontal rotating shaft of a rotating culture device,the rotating culture vessel characterized by comprising: one or moreinlets/outlets for supplying a cell and a liquid culture medium at ainitial stage and taking out a cultured cell at an appropriate positionon a flat cylindrical culture container; and at least one pair of asupply port and a discharge port for liquid culture medium exchange onan outer circumferential cylindrical face of the culture container, thepair of the supply port and the discharge port being positioned 180°opposite to each other, the discharge port having a center line passingthrough a rotating center, and the supply port having a center lineeccentrically positioned with respect to the rotating center.
 2. Therotating culture vessel according to claim 1, wherein a distance betweenthe center line of the supply port and the rotating center is 0.5r to0.9r where the culture container has a culture space having a radius ofr.
 3. The rotating culture vessel according to claim 1, wherein threepairs of the supply ports and the discharge ports are provided on theouter circumferential cylindrical face of the culture container, and thesupply ports and the discharge ports are provided at an equal angleinterval.
 4. The rotating culture vessel according to claim 1, whereineach of the supply port and the discharge port has a septum sealstructure, a supply syringe used for liquid culture medium exchange hasa leading end having an injection needle optionally passing through theseptum seal, a discharge syringe used for liquid culture medium exchangehas a leading end having an injection needle optionally passing throughthe septum seal, a new liquid culture medium is supplied from the supplysyringe while a used liquid culture medium is sucked with the dischargesyringe, and air is optionally bled with the discharge syringe.
 5. Therotating culture vessel according to claim 1, wherein the outercircumferential cylindrical face of the culture container includes acell suspension supply port for supplying a cell suspension composed ofa cell and a liquid culture medium, a front face orthogonal to therotating shaft of the culture container includes an air bleeding portand a cell discharge port, each of the cell suspension supply port andthe air bleeding port has a septum seal structure, and the celldischarge port has a rubber plug structure with a large opening.
 6. Therotating culture vessel according to claim 4, wherein each port havingthe septum seal structure includes an inlet flow path having a largediameter for accepting the injection needle behind the septum seal andan orifice flow path having a small diameter between the inlet flow pathand the culture space of the culture container.
 7. An automatic cellculture apparatus using the rotating culture vessel according to claim 1and used for three-dimensional culture of a cell in a nearlygravity-free microgravity environment, the automatic cell cultureapparatus characterized by comprising: a closed housing having an airconditioning function; an incubator box of a rotating culture device ina middle chamber of the closed housing; a cool supply box storing asupply syringe for supplying a liquid culture medium in a lower chamberof the closed housing; and a cool discharge box storing a dischargesyringe for collecting a liquid culture medium in an upper chamber ofthe closed housing; each of the incubator box, the cool supply box, andthe cool discharge box including a front face having an automaticallyoperated door, the rotating culture device having a shaft directionshifting means shifting a horizontal rotating shaft back and forth in ashaft direction, the horizontal rotating shaft being provided in theincubator box the rotating shaft having an end removably installed withthe rotating culture vessel, a front space of the cool supply boxincluding an XYZ-axis shifting mechanism for supply, the XYZ-axisshifting mechanism for supply including a movable part for supply drivenby the mechanism, the movable part for supply having a fixing chuck forholding the supply syringe upward and a push up means for pushing apiston up, a front space of the cool discharge box including an XYZ-axisshifting mechanism for discharge, the XYZ-axis shifting mechanism fordischarge including a movable part for discharge driven by themechanism, the movable part for discharge having a fixing chuck forholding the discharge syringe downward and a pull up means for pulling apiston up, each door being opened, the shaft direction shifting meansbeing driven to bring the rotating culture vessel from the incubator boxto a front space, the rotating culture vessel being stopped with thesupply port facing downward and the discharge port facing upward, theXYZ-axis shifting mechanism for supply and the fixing chuck being drivento take out the supply syringe from the cool supply box and to locatethe supply syringe below the rotating culture vessel, the XYZ-axisshifting mechanism for discharge and the fixing chuck being driven totake out the discharge syringe from the cool discharge box and to locatethe discharge syringe above the rotating culture vessel, the XYZ-axisshifting mechanism for supply being driven to airtightly connect thesupply syringe to the supply port, simultaneously, the XYZ-axis shiftingmechanism for discharge being driven to airtightly connect the dischargesyringe to the discharge port, and then the push up means and the pullup means being synchronously driven to exchange a liquid culture mediumin the rotating culture vessel.
 8. The automatic cell culture apparatusaccording to claim 7, wherein each of the cool supply box and the cooldischarge box includes a rotating revolver type stacker and a pluralityof holders around the stacker, the stacker has a vertical rotating shaftcontrolled with a stepping motor, and each holder elastically removablyholds the syringe in a transverse direction with the syringe in thevertical direction.
 9. The automatic cell culture apparatus according toclaim 7, wherein the rotating culture device includes a plurality of therotating shafts shifted back and forth by a common shaft directionshifting means, the rotating shafts are not overlapped in the verticaldirection, ends of the rotating shafts are displaced back and forth, andthe adjacent rotating culture vessels do not interfere with each otherin the case of the rotating culture vessels being installed to thecorresponding rotating shafts.
 10. The rotating culture vessel accordingto claim 2, wherein three pairs of the supply ports and the dischargeports are provided on the outer circumferential cylindrical face of theculture container, and the supply ports and the discharge ports areprovided at an equal angle interval.
 11. The rotating culture vesselaccording to claim 2, wherein each of the supply port and the dischargeport has a septum seal structure, a supply syringe used for liquidculture medium exchange has a leading end having an injection needleoptionally passing through the septum seal, a discharge syringe used forliquid culture medium exchange has a leading end having an injectionneedle optionally passing through the septum seal, a new liquid culturemedium is supplied from the supply syringe while a used liquid culturemedium is sucked with the discharge syringe, and air is optionally bledwith the discharge syringe.
 12. The rotating culture vessel according toclaim 2, wherein the outer circumferential cylindrical face of theculture container includes a cell suspension supply port for supplying acell suspension composed of a cell and a liquid culture medium, a frontface orthogonal to the rotating shaft of the culture container includesan air bleeding port and a cell discharge port, each of the cellsuspension supply port and the air bleeding port has a septum sealstructure, and the cell discharge port has a rubber plug structure witha large opening.
 13. An automatic cell culture apparatus using therotating culture vessel according to claim 2 and used forthree-dimensional culture of a cell in a nearly gravity-freemicrogravity environment, the automatic cell culture apparatuscharacterized by comprising: a closed housing having an air conditioningfunction; an incubator box of a rotating culture device in a middlechamber of the closed housing; a cool supply box storing a supplysyringe for supplying a liquid culture medium in a lower chamber of theclosed housing; and a cool discharge box storing a discharge syringe forcollecting a liquid culture medium in an upper chamber of the closedhousing; each of the incubator box, the cool supply box, and the cooldischarge box including a front face having an automatically operateddoor, the rotating culture device having a shaft direction shiftingmeans shifting a horizontal rotating shaft back and forth in a shaftdirection, the horizontal rotating shaft being provided in the incubatorbox the rotating shaft having an end removably installed with therotating culture vessel, a front space of the cool supply box includingan XYZ-axis shifting mechanism for supply, the XYZ-axis shiftingmechanism for supply including a movable part for supply driven by themechanism, the movable part for supply having a fixing chuck for holdingthe supply syringe upward and a push up means for pushing a piston up, afront space of the cool discharge box including an XYZ-axis shiftingmechanism for discharge, the XYZ-axis shifting mechanism for dischargeincluding a movable part for discharge driven by the mechanism, themovable part for discharge having a fixing chuck for holding thedischarge syringe downward and a pull up means for pulling a piston up,each door being opened, the shaft direction shifting means being drivento bring the rotating culture vessel from the incubator box to a frontspace, the rotating culture vessel being stopped with the supply portfacing downward and the discharge port facing upward, the XYZ-axisshifting mechanism for supply and the fixing chuck being driven to takeout the supply syringe from the cool supply box and to locate the supplysyringe below the rotating culture vessel, the XYZ-axis shiftingmechanism for discharge and the fixing chuck being driven to take outthe discharge syringe from the cool discharge box and to locate thedischarge syringe above the rotating culture vessel, the XYZ-axisshifting mechanism for supply being driven to airtightly connect thesupply syringe to the supply port, simultaneously, the XYZ-axis shiftingmechanism for discharge being driven to airtightly connect the dischargesyringe to the discharge port, and then the push up means and the pullup means being synchronously driven to exchange a liquid culture mediumin the rotating culture vessel.